1. Field of the Invention
The invention relates to oncogenes and to cancer diagnostics and therapeutics. More specifically, the present invention relates to amplified and/or overexpressed Cathepsin Z (CTSZ) and CD24 genes that are involved in certain types of cancers. The invention pertains to the amplified genes, their encoded proteins, and antibodies, inhibitors, activators and the like and their use in cancer diagnostics, vaccines, and anti-cancer therapy, including colon cancer, ovarian cancer and breast cancer.
2. Background of the Invention
Cancer and Gene Amplification:
Cancer is the second leading cause of death in the United States, after heart disease (Boring, et al., CA Cancer J. Clin., 43:7, 1993), and it develops in one in three Americans. One of every four Americans dies of cancer. Cancer features uncontrolled cellular growth, which results either in local invasion of normal tissue or systemic spread of the abnormal growth. A particular type of cancer or a particular stage of cancer development may involve both elements.
The division or growth of cells in various tissues functioning in a living body normally takes place in an orderly and controlled manner. This is enabled by a delicate growth control mechanism, which involves, among other things, contact, signaling, and other communication between neighboring cells. Growth signals, stimulatory or inhibitory, are routinely exchanged between cells in a functioning tissue. Cells normally do not divide in the absence of stimulatory signals, and will cease dividing when dominated by inhibitory signals. However, such signaling or communication becomes defective or completely breaks down in cancer cells. As a result, the cells continue to divide; they invade adjacent structures, break away from the original tumor mass, and establish new growth in other parts of the body. The latter progression to malignancy is referred to as “metastasis.”
Cancer generally refers to malignant tumors, rather than benign tumors. Benign tumor cells are similar to normal, surrounding cells. These types of tumors are almost always encapsulated in a fibrous capsule and do not have the potential to metastasize to other parts of the body. These tumors affect local organs but do not destroy them; they usually remain small without producing symptoms for many years. Treatment becomes necessary only when the tumors grow large enough to interfere with other organs. Malignant tumors, by contrast, grow faster than benign tumors, and they penetrate and destroy local tissues. Some malignant tumors may spread throughout the body via blood or the lymphatic system. The unpredictable and uncontrolled growth makes malignant cancers dangerous, and fatal in many cases. These tumors are not morphologically typical of the original tissue and are not encapsulated. Malignant tumors commonly recur after surgical removal.
Accordingly, treatment ordinarily is directed towards malignant cancers or malignant tumors. The intervention of malignant growth is most effective at the early stage of the cancer development. It is thus exceedingly important to discover sensitive markers for early signs of cancer formation and to identify potent growth suppression agents associated therewith. The development of such diagnostic and therapeutic agents involves an understanding of the genetic control mechanisms for cell division and differentiation, particularly in connection with tumorigenesis.
Cancer is caused by inherited or acquired mutations in cancer genes, which have normal cellular functions and which induce or otherwise contribute to cancer once mutated or expressed at an abnormal level. Certain well-studied tumors carry several different independently mutated genes, including activated oncogenes and inactivated tumor suppressor genes. Each of these mutations appears to be responsible for imparting some of the traits that, in aggregate, represent the full neoplastic phenotype (Land et al., Science, 222:771, 1983; Ruley, Nature, 4:602, 1983; Hunter, Cell, 64:249, 1991).
One such mutation is gene amplification. Gene amplification involves a chromosomal region bearing specific genes undergoing a relative increase in DNA copy number, thereby increasing the copies of any genes that are present. In general, gene amplification often results in increased levels of transcription and translation, producing higher amounts of the corresponding gene mRNA and protein. Amplification of genes causes deleterious effects, which contribute to cancer formation and proliferation (Lengauer et al. Nature, 396:643-649,1999).
It is commonly appreciated by cancer researchers that whole collections of genes are demonstrably overexpressed or differentially expressed in a variety of different types of tumor cells. Yet, only a very small number of these overexpressed genes are likely to be causally involved in the cancer phenotype. The remaining overexpressed genes likely are secondary consequences of more basic primary events, for example, overexpression of a cluster of genes, involved in DNA replication. On the other hand, gene amplification is established as an important genetic alteration in solid tumors (Knuutila et al., Am. J. Pathol., 152(5):1107-23, 1998; Knuutila et al., Cancer Genet. Cytogenet., 100(1):25-30, 1998).
The overexpression of certain well known genes, for example, c-myc, has been observed at fairly high levels in the absence of gene amplification (Yoshimoto et al., JPN J. Cancer Res., 77(6):540-5, 1986), although these genes are frequently amplified (Knuutila et al., Am. J. Pathol., 152(5):1107-23, 1998) and thereby activated. Such a characteristic is considered a hallmark of oncogenes. Overexpression in the absence of amplification may be caused by higher transcription efficiency in those situations. In the case of c-myc, for example, Yoshimoto et al. showed that its transcriptional rate was greatly increased in the tested tumor cell lines. The characteristics and interplay of overexpression and amplification of a gene in cancer tissues, therefore, provide significant indications of the gene's role in cancer development. That is, increased DNA copies of certain genes in tumors, along with and beyond its overexpression, may point to their functions in tumor formation and progression.
It must be remembered that overexpression and amplification are not the same phenomenon. Overexpression can be obtained from a single, unamplified gene, and an amplified gene does not always lead to greater expression levels of mRNA and protein. Thus, it is not possible to predict whether one phenomenon will result in, or is related to, the other. However, in situations where both amplification of a gene and overexpression of the gene product occur in cells or tissues that are in a precancerous or cancerous state, then that gene and its product present both a diagnostic target and a therapeutic opportunity for intervention. Because some genes are sometimes amplified as a consequence of their location next to a true oncogene, it is also beneficial to determine the DNA copy number of nearby genes in a panel of tumors so that amplified genes that are in the epicenter of the amplification unit can be distinguished from amplified genes that are occasionally amplified due to their proximity to another, more relevant amplified gene.
Thus, discovery and characterization of amplified cancer genes, along with and in addition to their features of overexpression or differential expression, will be a promising avenue that leads to novel targets for diagnostic, vaccines, and therapeutic applications.
Additionally, the completion of the working drafts of the human genome and the paralleled advances in genomics technologies offer new promises in the identification of effective cancer markers and the anti-cancer agents. The high-throughput microarray detection and screening technology, computer-empowered genetics and genomics analysis tools, and multi-platform functional genomics and proteomics validation systems, all assist in applications in cancer research and findings. With the advent of modern sequencing technologies and genomic analyses, many unknown genes and genes with unknown or partially known functions can be revealed.
Homo sapiens CTSZ: Cysteine proteases belonging to the papain family represent a major component of the lysosomal proteolytic system and play an essential role in protein degradation and turnover. To date, ten human cysteine proteases of the papain family have been isolated and characterized at the amino acid sequence level: cathepsin B, cathepsin L, cathepsin H, cathepsin S, cathepsin C, cathepsin O, cathepsin K, cathepsin W, cathepsin L2 and cathepsin Z (CTSZ). Existence of additional cysteine proteases including cathepsins M, N, and T, have been documented. These proteases have been originally identified because of their degrading activity on specific substrates such as aldolase, collagen, proinsulin, or tyrosine aminotransferase (Santamaria, et al., Cancer Res, 58:624-1630, 1998).
CTSZ is also named as cathepsin X or cathepsin P. A full-length cDNA for CTSZ was first cloned in 1998 by Santamaria et al. from a human brain cDNA library (J Biol Chem, 273(27):16816-16823, 1998). The CTSZ DNA of 1501 nucleotides encodes a protein of 303 amino acids. The amino acid sequence encoded by the DNA for CTSZ shows a high degree of identity to cysteine proteases. The human CTSZ gene maps to chromosome 20q13, a location that differs from all cysteine protease genes. On the basis of a series of distinctive structural features, including diverse peptide insertions and an unusual short propeptide, together with its unique chromosomal location among cysteine proteases, CTSZ is regarded as the first representative of a novel subfamily of this class of proteolytic enzymes. Cathepsin Z shares protein sequence identity with other human cysteine proteases of the papain family, including 34% with cathepsin C and 26% with cathepsin B. Cathepsin B at 8p22 is amplified in esophageal adenocarcinoma and overexpressed in esophageal adenocarcinoma, lung, prostate, colon, breast and stomach tumors.
CTSZ is widely expressed in human tissues and therefore the enzyme could be involved in the normal intracellular protein degradation taking place in all cell types. CTSZ is also reported ubiquitously distributed in cancer cell lines and in primary tumors. Recombinant CTSZ exhibited enzymatic activity with substrate specificity and sensitivity toward inhibitors characteristic of cysteine proteases. Therefore, CTSZ has the potential of invasion through its protease activity, and participation in tumor progression like other cathepsins (see WO 99/31256; U.S. Pat. Nos. 5,783,434; 5,849,711; 5,858,982; JP2000-50885).
Homo sapiens CD24: Homo sapiens CD24 antigen (small cell lung carcinoma cluster 4 antigen) (CD24) is located on the human chromosome 6q21. CD24 is a cell surface antigen, a sialoglycoprotein, that is anchored to the cell surface by a glycosyl phosphatidylinositol linkage. It is expressed in many B-lineage cells and on mature granulocytes. Studies with monoclonal antibodies, however, indicate that most other hematopoietic cells, including T cells, monocytes, red blood cells, and platelets, seem not to express the CD24 antigen. The CD24 DNA is approximately 2.1 kb in length with a coding region of 243 (see SEQ ID NO:4, encoding region 57-299) nucleotides (see SEQ ID NO:6), which encodes a protein of 80 amino acids (see SEQ ID NO:5) (Huang et al., Cancer Res, 55(20):4717-21, 1995; Jackson et al., Cancer Res, 52(19):5264-70, 1992).
CD24 has been identified as a ligand for P-selectin in both mouse and human cells. It has been reported that the P-selectin-CD24 binding pathway is important for the binding of the breast carcinoma cell line KS to platelets and the rolling of these cells on endothelial P-selectin (Fogel et al., Cancer Lett, 143(1):87-94, 1999; Frienderichs et al., Cancer Res, 60:6714-6722, 2000). Since CD24 binds P-selectin that is found on blood vessels, it has been speculated that its expression could help the cells to reach blood vessels (Aigner et al., Blood, 89(9):3385-95, 1997). This, however, was highly speculative and the investigators failed to show that-CD24 expression is functionally important in tumor formation.
CD24 has been suggested as a cellular marker (U.S. Pat. Nos. 5,804,177; 6,146,628) and also as a marker in breast and lung carcinomas (Fogel et al., Cancer Lett, 143(1):87-94, 1999; Jackson et al., Cancer Res, 52(19):5264-70, 1992). Anti-CD24 antibody also has been suggested to treat B-cell disorder after transplantation (Benkerrou et al., Blood, 92(9):3137-3147, 1998). However, its role in tumorogenesis, amplification and overexpression of the CD24 gene in cancers has not been discussed.
Additionally, the possibility to treat tumors with antibodies that block the oncogenic function of CD24, as opposed to antibodies that bind to tumor cells expressing CD24 and thereby mediate tumor-cell killing by mechanisms unrelated to the disclosed oncogenic CD24 function, was not known until the present invention. Therefore, there is a need in the art for an understanding of CTSZ and CD24 gene regulation. Understanding the physiological role of human CTSZ and CD24 genes will facilitate early diagnosis of abnormalities associated therewith and lead to appropriate therapies to treat such abnormalities.